RT Journal Article SR Electronic T1 Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.03.25.436878 DO 10.1101/2021.03.25.436878 A1 Guillaume A. Petit A1 Biswarajan Mohanty A1 Róisín M. McMahon A1 Stefan Nebl A1 David H. Hilko A1 Karyn L. Wilde A1 Martin J. Scanlon A1 Jennifer L. Martin A1 Maria A. Halili YR 2021 UL http://biorxiv.org/content/early/2021/03/26/2021.03.25.436878.abstract AB DiSulfide Bond forming proteins (DSB) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide bond protein A (DsbA) catalyzes the formation of disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, we identified two fragments - 1 (bromophenoxy propanamide) and 2 (4-methoxy-N-phenylbenzenesulfonamide), that bind to the DsbA from the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis. Crystal structures of the oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 suggests that both fragments bind to a hydrophobic pocket that is formed by a change in the side chain orientation of tyrosine 110. This conformational change opens a “cryptic” pocket that is not evident in the apo-protein structure. This binding location was supported by 2D-NMR studies which identified a chemical shift perturbation of the tyrosine 110 backbone amide resonance of more than 0.05 ppm upon addition of 2 mM of fragment 1 and over 0.04 ppm upon addition of 1 mM of fragment 2. Although binding was detected by both X-ray crystallography and NMR, the binding affinity (KD) for both fragments was low (above 2 mM), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the modelled crystal structures which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have high binding affinity due to their size and the relatively small surface area that can be involved in intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. Identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.Synopsis Describes the binding properties of two drug-like fragments to a conformationally dynamic site in the disulfide-bond forming protein A from Burkholderia pseudomallei.Competing Interest StatementThe authors have declared no competing interest.